This invention relates to bacterial adhesin proteins and active fragments thereof for use in vaccine compositions for the prevention, diagnosis and treatment of bacterial induced diseases such as those of the urinary tract, especially to the use of such adhesins as immunogenic agents in humans and animals to stimulate an immune response.
Urinary tract infections (herein, xe2x80x9cUTIxe2x80x9d) present a disease process that is mediated by the attachment of bacteria to cells. Escherichia coli is the most common pathogen of the urinary tract, accounting for more than 85% of cases of asymptomatic bacteriuria, acute cystitis and acute pyelonephritis, as well as greater than 60% of recurrent cystitis, and at least 35% of recurrent pyelonephritis infections. Furthermore, approximately 25%-30% of women experience a recurrent E. coli urinary tract infection within the first 12 months following an initial infection but after a second or third infection the rate of recurrence increases to 60%-75%. Given the high incidence, continued persistence, and significant expense associated with E. coli urinary tract infections, there is a need for a prophylactic vaccine to reduce susceptibility to this disease.
While many factors contribute to the acquisition and progression of E. coli urinary tract infections, it is generally accepted that colonization of the urinary epithelium is a required step in the infection process. In a typical course of E. coli urinary tract infection, bacteria originate from the bowel, ascend into the bladder, and adhere to the bladder mucosa where they multiply and establish an infection (cystitis) before ascending into the ureters and kidneys. Disruption or prevention of pilus-mediated attachment of E. coli to urinary epithelia may prevent or retard the development of urinary tract infections. In this regard, a number of studies have pointed to a role for pili in mediating attachment to host uroepithelial cells.
To initiate infection bacterial pathogens must first be able to colonize an appropriate target tissue of the host. For many pathogens this tissue is located at a mucosal surface. Colonization begins with the attachment of the bacterium to receptors expressed by cells forming the lining of the mucosa. Attachment is mediated via proteins on the bacterium that bind specifically to cellular receptors. These proteins, or adhesins, are expressed either directly on the surface of the bacterium, or more typically, as components of elongated rod-like protein structures called pili, fimbriae or fibrillae.
Type 1 pili are thought to be important in initiating colonization of the bladder and inducing cystitis, whereas P pili are thought to play a role in ascending infections and the ensuing pyelonephritis.
Such pili are heteropolymeric structures that are composed of several different structural proteins required for pilus assembly. Two types of pili are of particular interest: P pili and type 1 pili. P pili-carrying bacteria recognize and bind to the gal-(xcex11-4)gal moiety present in the globoseries of glycolipids on kidney cells in mammals. Type 1 pili-carrying bacteria recognize and bind to D-mannose in glycolipids and glycoproteins of bladder epithelial cells.
FimH is the D-mannose-binding adhesin that promotes attachment of type 1 piliated bacteria to host cells via mannose-containing glycoproteins on eukaryotic cell surfaces. FimC is its periplasmic chaperone protein.
In this specification, the terms xe2x80x9cpilixe2x80x9d, xe2x80x9cfimbriae,xe2x80x9d and xe2x80x9cfibrillaexe2x80x9d are used to refer to heteropolymeric protein structures located on the extracellular surface of bacteria, most commonly gram-negative bacteria. Typically these structures are anchored in the outer membrane. Throughout this specification the terms pilus, pili, fimbriae, and fibrilla will be used interchangeably.
As used herein, the term xe2x80x9cperiplasmic chaperonexe2x80x9d is defined as a protein localized in the periplasm of bacteria that is capable of forming complexes with a variety of chaperone-binding proteins via recognition of a common binding epitope (or epitopes). Chaperones serve as templates upon which proteins exported from the bacterial cell into the periplasm fold into their native conformations. Association of the chaperone-binding protein with the chaperone also serves to protect the binding proteins from degradation by proteases localized within the periplasm, increases their solubility in aqueous solution, and leads to their sequentially correct incorporation into an assembling pilus.
Chaperone proteins are a class of proteins in gram-negative bacteria that are involved in the assembly of pili by mediating such assembly, but are not incorporated into the structure. FimC is the periplasmic chaperone protein that mediates assembly of type 1 pili in bacteria.
It has recently been reported that such chaperones can direct formation of the appropriate native structure of the corresponding adhesin or pilin by inserting a specific fold of the chaperone protein in place of a missing domain or helical strand of the chaperone or pilin. Thus, FimH proteins tend to have their native structure in the presence of such a chaperone but not in its absence. [see: Choudhury et al, X-ray Structure of the FimC-FimH Chaperone-Adhesin Complex from Uropathogenic E. coli, Science 285, 1061 (1999); Sauer et al, Structural Basis of Chaperone Function and Pilus Biogenesis, Science 285, 1058 (1999)] In addition, recent publications have indicated that the required chaperone strand can be inserted into the adhesin or pilin protein, such as FimH, to provide the missing structure and produce the correct native structure; Barnhart, M. M. et al., PapD-like Chaperones Provide the Missing Information for Folding of Pilin Proteins, Proc. Natl. Acad. Sci. (USA), 10.1073/pnas.130183897 (published online Jun. 20, 2000).
Antibodies directed against purified whole type 1 or P pili protect against cystitis and pyelonephritis, respectively, in both murine and primate models for these diseases. See: Abraham et al., Infect Immun. 48:625 (1985), Roberts et al., Proc. Natl. Acad. Sci. (USA) 91:11889 (1994), O""Hantey et al., J. Clin. Invest. 75: 347 (1985). However, such protection is limited to either homologous E. coli strains from which the pili used as immunogens were derived, or to a small subset of serologically cross-reactive heterologous strains. Therefore, vaccines composed predominantly of the major structural proteins of pili (i.e., PapA or FimA) appear to be of limited value because antibodies developed against these highly variable proteins are specific for the strains used for immunization.
Furthermore, antibodies to FimH have been found to be protective. Barnhart, M. M. et al., PapD-like Chaperones Provide the Missing Information for Folding of Pilin Proteins, Proc. Natl. Acad. Sci. (USA), 10.1073/pnas.130183897 (published online Jun. 20, 2000).
Recently, Sokurenko et al [see: J. Bacteriol. 177, 3680-86 (1995)] have found that quantitative variations in mannose-sensitive adhesion of E. coli are due primarily to structural differences in the FimH adhesin. Further research has shown that the ability of the FimH lectins to interact with monomannosyl residues strongly correlates with their ability to mediate E. coli adhesion to uroepithelial cells so that certain phenotypic variants of type 1 fimbriae may contribute more than others to the virulence of E. coli in the urinary tract. [Sokurenko et al, J. Biol. Chem. 272, 17880-17886 (1997)]. Heretofore, random point mutations in FimH genes that increase binding of the adhesin to mono-mannose residues (structures abundant in the oligosaccharide moieties of urothelial glycoproteins) have been found to confer increased virulence in the mouse urinary tract. [See: Sokurenko et al, Proc. Natl. Acad. Sci. USA 95, 8922-8926 (1998)]
In one aspect, the present invention relates to immunogenic polypeptides derived from different strains of the bacterium Escherichia coli (E. coli) which differ from each other in one or more amino acid residues and that induce immunological responses that lead to protection of an animal, especially a human patient, following vaccination with said polypeptides.
It is an object of the present invention to provide immunogenic FimH polypeptides useful as components of vaccines for the prevention and treatment of infections caused by E. coli, in particular, urinary tract infections. In specific embodiments, the polypeptides of the present invention have the amino acid sequences shown in FIG. 2. Such vaccine compositions comprising the novel polypeptides disclosed herein are useful in vaccination and treatment of urinary tract infections, especially those caused by E. coli. In one embodiment, vaccine compositions according to the invention comprise the polypeptides of FIG. 2 (or truncated segments of the sequences of FIG. 2 which truncates have mannose-binding ability and can serve as vaccines), as stabilized structures in the form of complexes with a chaperone, such as FimC, or with a portion of the sequence of FimC which portion serves to stabilize the structure of the FimH adhesin. A particular embodiment employs the consensus sequence of FIG. 2 (SEQ ID NO: 55).
It is another object of the present invention to provide antibodies, including both polyclonal and monoclonal antibodies, with specificity for the novel polypeptides disclosed herein and whose amino acid sequences are shown in FIG. 2.
It is a still further object of the present invention to provide methods of prophylaxis for the prevention of urinary tract infections using the vaccine compositions disclosed herein.
It is a yet still further object of the present invention to provide methods of treatment of diseases of the urinary tract comprising the use of vaccine compositions as disclosed herein and the use of antibodies generated against such vaccine compositions, and the polypeptides contained therein.
It is another object of the present invention to provide a novel method of preparing polypeptides from recombinant cells using a vector comprising the plasmid of FIGS. 3 through 6. In specific embodiments, the polypeptides comprise the amino acid sequences of FIG. 2. In one embodiment, this process and plasmid can be used to prepare polypeptides that comprise a bacterial chaperone, such as FimC, fused to a bacterial adhesin, such as FimH or any of the polypeptides presented in FIG. 2, including any consensus sequence derived therefrom.